Human peripheral and gastric lymphocyte responses to Helicobacter pylori NapA and AphC differ in infected and uninfected individuals

Abstract

Background: In this study, we identify the nature of the immunological response of human peripheral blood mononuclear cells (PBMC) and lamina propria gastric lymphocytes (LPL) to two Helicobacter pylori antigens, the neutrophil activating protein (NapA) and alkyl hydroperoxide reductase (AphC). These antigens were identified and selected for study based on the observation that serological recognition of these proteins was associated with H pylori negative status in humans.

Aims: The aim was to study the serological, proliferative, and cytokine responses of PBMC and LPL, obtained from H pylori infected and uninfected individuals, to these antigens.

Methods: Patient serum, PBMC, and LPL were used to determine antibody isotype, and proliferative and cytokine responses to recombinant forms of NapA and AphC using western blotting and ELISA.

Results: Western blotting revealed antibody reactivity to recombinant NapA and AphC among the H pylori negative population studied. Both the proliferative and interferon gamma responses of PBMC and LPL to NapA and AphC were significantly higher in H pylori negative compared with H pylori positive subjects. Analysis of the IgG subclass profiles to both antigens revealed a T helper 1 associated IgG3 antibody response in uninfected individuals. However, interleukin 10 production was greater in H pylori positive individuals in response to these antigens.

Conclusions: Taken together these data are consistent with an immune response to these antigens skewed towards a T helper 1 response in the uninfected cohort.

Figure 1

Serum samples screened for the presence of anti-Helicobacter pylori IgG antibodies. Western blots of whole H pylori (50 μg/lane) probed with serum obtained from H pylori infected (A) and H pylori uninfected (B) cohorts are shown. All sera were diluted 1:100 in phosphate buffered saline containing fat free dried skimmed milk (5% w/v). Each track represents an individual strip of PVDF membrane which had been incubated with a different serum sample. Blots were developed by enhanced chemiluminescence.

Figure 2

Adsorption of sera from infected and uninfected seropositive subjects. Serum (40 μl) from either uninfected (sera 1, 2, 3) or infected (serum 4) individuals was either untreated (C) or adsorbed with sonicates of H pylori (Hp), C jejuni (Cj), E aerogenes (Ea), S typhimurium (St), or Y pseudotuberculosis (Yp) prior to probing blots of whole Helicobacter pylori (NCTC26695) with each serum sample. Primary IgG was detected with horseradish peroxidase conjugated rabbit antihuman IgG (1/3000) and developed by enhanced chemiluminescence.

Figure 3

C jejuni and E coli antigens recognised by anti-Helicobacter pylori antiserum and elimination of cross reactivity by adsorption with H pylori. (A) Western blot of H pylori (lane 1), C jejuni (lane 2), and E coli (lane 3) probed with rabbit anti-H pylori antiserum. A sonicate of each bacterium (50 μg) was subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting, as described in the methods section. (B) Effect of adsorbing serum obtained from a H pylori infected subject (lanes 1–3) and a subject negative for H pylori (lanes 4–6) with E coli (lanes 1 and 4), H pylori (lanes 2 and 5), or C jejuni (lanes 3 and 6).

Figure 4

Immunoreactivity of neutrophil activating protein (NapA) and alkyl hydroperoxide reductase (AphC) with sera from uninfected seropositive subjects. Untreated sera (1/50) were used to probe blots of recombinant NapA (A, B) or AphC (E). Selected sera from these individuals were also adsorbed with the bacterial sonicates described in the legend to fig 2 ▶, prior to probing blots of NapA (C, three sera and D, three sera) or AphC (F, four sera). The blots were processed as described in fig 2 ▶.

Figure 5

Total IgG and IgG subclass responses to neutrophil activating protein (NapA) and alkyl hydroperoxide reductase (AphC). Total IgG response and the various IgG subclass responses to NapA (A) and AphC (B) in serum obtained from Helicobacter pylori positive and H pylori negative individuals are shown. IgG levels were determined by ELISA, as described in the methods section and the absorbance axes represent readings obtained at 450 nm for total IgG and 405 nm for the IgG subclasses.

Figure 6

Proliferative responses of peripheral blood mononuclear cells (PBMC) and lamina propria lymphocytes (LPL) to neutrophil activating protein (NapA) and alkyl hydroperoxide reductase (AphC). The proliferative responses of PBMC (A) and LPL (B) to Helicobacter pylori sonicate (HPS) (3 μg/ml for PBMC and 300 μg/ml for LPL), NapA (1 μg/ml), AphC (1 μg/ml), and recombinant urease B subunit (rUreB) (1 μg/ml) are shown for both H pylori positive H pylori uninfected subjects. Results are expressed as [³H]-thymidine incorporation (cpm) into PBMC (2×10⁶/ml) and LPL (4×10⁵/ml) cultured for three days in the presence of the indicated antigens. All samples were measured in triplicate.

Figure 7

Interferon γ (IFN-γ) production by peripheral blood mononuclear cells (PBMC) and lamina propria lymphocytes (LPL) in response to neutrophil activating protein (NapA) and alkyl hydroperoxide reductase (AphC). IFN-γ production by PBMC (A) and LPL (B) obtained from Helicobacter pylori positive or H pylori negative individuals in response to the indicated antigens. All antigens were used at 1 μg/ml. Supernatants were collected from cultured PBMC and LPL after 72 hours and stored at −80°C. IFN-γ was measured in the supernatant by ELISA. All samples were measured in duplicate. Results are expressed as mean (SEM). rUreB, recombinant urease B subunit; HPS, H pylori sonicate.

Figure 8

Interleukin 10 (IL-10) production by peripheral blood mononuclear cells (PBMC) and lamina propria lymphocytes (LPL) in response to neutrophil activating protein (NapA) and alkyl hydroperoxide reductase (AphC). Levels of IL-10 produced by PBMC (A) and LPL (B) from H pylori positive and H pylori negative subjects in response to the antigens indicated are shown. All antigens were used at 1 μg/ml. Supernatants were collected from cultured PBMC and LPL after 72 hours and stored at −80°C. IL-10 was measured in the supernatant by ELISA. All samples were determined in duplicate. Results were expressed as mean (SEM). rUreB, recombinant urease B subunit; HPS, H pylori sonicate.